Search

Dry eye disease (DED) is an inflammatory disorder in which CD4 + T cells play a significant role in its pathogenesis. A CD4 + T cell subset termed granulocyte-macrophage colony-stimulating factor-producing T helper (ThGM) cells would contribute to DED pathogenesis. However, the mechanisms by which the activity of ThGM cells is modulated are not thoroughly understood. In this research, we characterized the effects of neurokinin 1 receptor (NK1R) and neurokinin 2 receptor (NK2R) on ThGM cells and T helper 1 (Th1) cells in a murine DED model. We found that ThGM cells expressed NK1R and NK2R, whereas Th1 cells predominantly expressed NK1R. Furthermore, substance P and neurokinin A (NKA), the ligands of NK1R and NK2R, were upregulated in post-DED LNs and conjunctivae. Substance P significantly promoted granulocyte-macrophage colony-stimulating factor (GM-CSF) expression while mildly upregulating the expression of interferon-gamma (IFN-γ) and interleukin 2 (IL-2) in ThGM cells. By contrast, NKA did not change GM-CSF expression but significantly increased IFN-γ expression in ThGM cells. Importantly, the adoptive transfer of NK1R-expressing ThGM cells significantly exacerbated DED, whereas the transfer of NK1R-knockdown ThGM cells weakly aggravated DED. NK2R knockdown in ThGM cells did not affect DED progression. In conclusion, this study identifies the substance P-NK1R axis as a novel mechanism that reinforces the pathogenicity of ThGM cells in DED., (© 2025 The Scandinavian Foundation for Immunology.)

The combination of decreasing food intake and increasing energy expenditure represents a powerful strategy for counteracting cardiometabolic diseases such as obesity and type 2 diabetes 1 . Yet current pharmacological approaches require conjugation of multiple receptor agonists to achieve both effects 2-4 , and so far, no safe energy-expending option has reached the clinic. Here we show that activation of neurokinin 2 receptor (NK2R) is sufficient to suppress appetite centrally and increase energy expenditure peripherally. We focused on NK2R after revealing its genetic links to obesity and glucose control. However, therapeutically exploiting NK2R signalling has previously been unattainable because its endogenous ligand, neurokinin A, is short-lived and lacks receptor specificity 5,6 . Therefore, we developed selective, long-acting NK2R agonists with potential for once-weekly administration in humans. In mice, these agonists elicit weight loss by inducing energy expenditure and non-aversive appetite suppression that circumvents canonical leptin signalling. Additionally, a hyperinsulinaemic-euglycaemic clamp reveals that NK2R agonism acutely enhances insulin sensitization. In diabetic, obese macaques, NK2R activation significantly decreases body weight, blood glucose, triglycerides and cholesterol, and ameliorates insulin resistance. These findings identify a single receptor target that leverages both energy-expending and appetite-suppressing programmes to improve energy homeostasis and reverse cardiometabolic dysfunction across species., Competing Interests: Competing interests: T.M., J.H.E., J.B.H., D.P.C., M.B.F.G., M.T.-C., T.W.S. and Z.G.-H. work or have worked in some capacity for Embark Laboratories ApS, a company developing therapeutics for the treatment of T2D and obesity. P.K. is a consultant for Embark Laboratories ApS. The use of and chemical composition for NK2R agonists are patented by the University of Copenhagen and Embark Laboratories ApS, respectively. R.J.S. has received research support from Novo Nordisk, Fractyl, AstraZeneca, Congruence Therapeutics, Eli Lilly, Bullfrog AI, Glycsend Therapeutics and Amgen. R.J.S. has served as a paid consultant for Novo Nordisk, Eli Lilly, CinRx, Fractyl, Structure Therapeutics, Crinetics and Congruence Therapeutics. R.J.S. has equity in Calibrate, Rewind and Levator Therapeutics. The other authors declare no competing interests., (© 2024. The Author(s).)

Neurokinin/tachykinin receptors are classified as the G-protein coupled receptor superfamily. The neurokinin 2 receptor (NK2R) is widely expressed in different tissues. NK2R is associated with a range of biological events, such as inflammation, smooth muscle contraction, intestinal motor functions, and asthma. Despite these diverse activities, no approved drugs targeting NK2R have been developed yet. Our study focuses on finding potential inhibitors for NK2R using virtual screening, molecular docking, and ADME (absorption, distribution, metabolism, and excretion) approaches. We used a homology modeling approach and AlphaFold DB to obtain the three-dimensional structure of mouse and human NK2R proteins, respectively. The homology model of NK2R was predicted using MODELLER v10.3 and further refined and validated using the 3Drefine tool and RAMPAGE server, respectively. Molecular docking was performed using a library of 910 structurally similar molecules to four NK1R antagonists: aprepitant, casopitant, fosaprepitant, and rolapitant. Molecular docking revealed six small molecules that displayed high Chemscore fitness scores, and binding energies with desirable ligand-NK2R interactions. The evaluation of the in silico ADME profile, solubility, and permeability of the ligand molecules has revealed that the small molecules are potentially nontoxic and have the chance of exhibiting biological activity after oral administration. Further experimental studies (in vitro and in vivo assays) are required to evaluate the effectiveness of these inhibitors as therapeutic targets., (© 2023 International Union of Biochemistry and Molecular Biology, Inc.)

The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P and neurokinin A, respectively, and have been targets for drug development. Despite sharing a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 that helps direct biological function, the peptide ligands exhibit some degree of cross-reactivity toward each other's non-natural receptor. Here, we investigate the detailed structure-activity relationships of the ligand-bound receptor complexes that underlie both potent activation by the natural ligand and cross-reactivity. We find that the specificity and cross-reactivity of the peptide ligands can be explained by the interactions between the amino acids preceding the FxGLM consensus motif of the bound peptide ligand and two regions of the receptor: the β-hairpin of the extracellular loop 2 (ECL2) and a N-terminal segment leading into transmembrane helix 1. Positively charged sidechains of the ECL2 (R177 of NK1R and K180 of NK2R) are seen to play a vital role in the interaction. The N-terminal positions 1 to 3 of the peptide ligand are entirely dispensable. Mutated and chimeric receptor and ligand constructs neatly swap around ligand specificity as expected, validating the structure-activity hypotheses presented. These findings will help in developing improved agonists or antagonists for NK1R and NK2R., Competing Interests: Conflict of interest D. P. C. and J. B. H. are employees of Embark Biotech ApS. All other authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Exposure to aerosolized citric acid (CA, 150 mM) and prostaglandin E 2 (PGE 2 , 0.43 mM) for 10 min in guinea pigs reportedly produces the distinct cough patterns (Type I vs. II) and ventilatory responses (long-lasting hyperventilation vs. brief tachypnea) even though triggering the same cough numbers. Type I and II coughs are primarily mediated by activation of TRPV1 and EP3 receptors (a PGE 2 receptor) of vagal C-fibers respectively. Substance P (SP) and neurokinin A (NKA) released by vagal pulmonary sensory fibers peripherally are capable of affecting CA-induced cough and ventilation via preferentially activating neurokinin 1 and 2 receptors (NK 1 R and NK 2 R) respectively. This study aimed to define the impacts of CA- and PGE 2 -exposure on pulmonary SP and NKA levels and the roles of NK 1 R and NK 2 R in modulating CA- and PGE 2 -evoked cough and ventilatory responses. In unanesthetized guinea pigs, we determined: (1) pulmonary SP and NKA contents induced by the CA- or PGE 2 -exposure; (2) effects of CP-99994 and SR-48968 (a NK 1 R and a NK 2 R antagonist respectively) given by intraperitoneal injection (IP) or aerosol inhalation (IH) on the CA- and PGE 2 -evoked cough and ventilatory responses; and (3) immunocytochemical expressions of NK 1 R/NK 2 R in vagal C-neurons labeled by TRPV1 or EP3 receptors. We found that CA- and PGE 2 -exposure evoked Type I and II cough respectively associated with different degrees of increases in pulmonary SP and NKA. Applications of CP-99994 and SR-48968 via IP and IH efficiently suppressed the cough responses to CA with less impact on the cough response to PGE 2 . These antagonists inhibited or blocked the ventilatory response to CA and caused hypoventilation in response to PGE 2 . Moreover, NK 1 R and NK 2 R were always co-expressed in vagal C-neurons labeled by TRPV1 or EP3 receptors. These results suggest that SP and NKA endogenously released by CA- and PGE 2 -exposure play important roles in generating the cough and ventilatory responses to CA and PGE 2 , at least in part, via activation of NK 1 R and NK 2 R expressed in vagal C-neurons (pulmonary C-neurons)., Competing Interests: Conflict of interest All authors declare no conflict of interest, (Copyright © 2022. Published by Elsevier B.V.)

Neurokinin 2 receptor (NK2R), a G protein-coupled receptor for neurokinin A (NKA), a tachykinin family member, regulates various physiological functions including pain response, relaxation of smooth muscle, dilation of blood vessels, and vascular permeability. However, the precise role and regulation of NK2R expression in cancer cells have not been fully elucidated. In this study, we found that high NK2R gene expression was correlated with the poor survival of colorectal cancer patients, and Interferon (IFN-α/β) stimulation significantly enhanced NK2R gene expression level of colon cancer cells in a Janus kinas 1/2 (JAK 1/2)-dependent manner. NKA stimulation augmented viability/proliferation and phosphorylation of Extracellular-signal-regulated kinase 1/2 (ERK1/2) levels of IFN-α/β-treated colon cancer cells and NK2R blockade by using a selective antagonist reduced the proliferation in vitro. Administration of an NK2R antagonist alone or combined with polyinosinic-polycytidylic acid, a synthetic analog of double-stranded RNA, to CT26-bearing mice significantly suppressed tumorigenesis. NK2R-overexpressing CT26 cells showed enhanced tumorigenesis and metastatic colonization in both lung and liver after the inoculation into mice. These findings indicate that IFN-α/β-mediated NK2R expression is related to the malignancy of colon cancer cells, suggesting that NK2R blockade may be a promising strategy for colon cancers., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Substance P (SP), a product of the tachykinin 1 (Tac1) gene, is expressed in many hypothalamic neurons. Its wake-promoting potential could be mediated through histaminergic (HA) neurons of the tuberomamillary nucleus (TMN), where functional expression of neurokinin receptors (NKRs) waits to be characterized. As in the process of nociception in the peripheral nervous system (PNS) capsaicin-receptor (transient potential vanilloid 1: TRPV1) signalling is amplified by local release of histamine and SP, we tested the involvement of tachykinins in the capsaicin-induced long-lasting enhancement (LLEcaps) of HA neurons firing by investigating selective neurokinin receptor ligands in the hypothalamic mouse brain slice preparation using patch-clamp recordings in cell-attached mode combined with single-cell RT-PCR. We report that the majority of HA neurons respond to SP (EC50 3 nM), express the SP precursor tachykinin 1 (Tac1) gene and at least one of the neurokinin receptors. Responses to selective agonists of three known neurokinin receptors were sensitive to corresponding antagonists. LLEcaps was significantly impaired by the neurokinin receptor antagonists, indicating that in hypothalamus, as in the PNS, release of tachykinins downstream to TRPV1 activation is able to boost the release of histamine. The excitatory action of SP on histaminergic neurons adds another pathway to the noradrenergic and orexinergic ones to synergistically enhance cortical arousal. We show NK1R to play a prominent role on HA neurons and thus the control of wakefulness., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Substance P (SP) and neurokinin A (NKA), encoded by TAC1 / Tac1 gene are members of the tachykinin family, which exert their neuromodulatory roles in vertebrate reproduction. In mammals, SP and NKA have been shown to regulate gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion via kisspeptin neurons. On the other hand, the role of SP/NKA in the regulation of reproduction in non-mammalian vertebrates is not well known. In the present study, we first localized expression of tac1 mRNA in the brain of male and female zebrafish, Danio rerio . Next, using an antibody against zebrafish tachykinin1 (Tac1), we examined the neural association of SP/NKA neural processes with GnRH3 neurons, and with kisspeptin ( kiss2 ) neurons, in the brains of male and female zebrafish. In situ hybridization showed an apparent male-dominant tac1 expression in the ventral telencephalic area, the anterior and posterior parts of the parvocellular preoptic nucleus, and the suprachiasmatic nucleus. On the other hand, there was female-dominant tac1 expression in the ventral periventricular hypothalamus. Confocal images of double-labeled zebrafish Tac1 and GnRH3 showed associations between Tac1-immunoreactive processes and GnRH3 neurons in the ventral telencephalic area. In contrast, there was no apparent proximity of Tac1 processes to kiss2 mRNA-expressing neurons in the hypothalamus. Lastly, to elucidate possible direct action of SP/NKA on GnRH3 or Kiss2 neurons, expression of SP/NKA receptor, tacr1a mRNA was examined in regions containing GnRH3 or Kiss2 neurons by in situ hybridization. Expression of tacr1a mRNA was seen in several brain regions including the olfactory bulb, preoptic area and hypothalamus, where GnRH3 and Kiss2 cells are present. These results suggest that unlike in mammals, Tac1 may be involved in male reproductive functions via direct action on GnRH3 neurons but independent of kisspeptin in the zebrafish., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ogawa, Ramadasan, Anthonysamy and Parhar.)

There is increasing focus on the involvement of tachykinins in immune and inflammatory responses. Hemokinin-1 (HK-1) is a recently identified tachykinin that originates primarily from immune cells, and has structural similarities to substance P (SP), found mainly in neurons. However, there are species differences in HK-1, and the role of HK-1 in humans, particularly the intestine, has received minimal attention. The aim of this study was to investigate the inflammatory role of human HK-1 in the human colon. The effects of HK-1 and SP were compared on the production of multiple inflammatory cytokines and chemokines from human colonic mucosal explants. Data generated by Procarta multiplex assay and QuantiGene assay demonstrated that 4 h incubation with HK-1 (0.1 μM) significantly stimulated transcript expression and release of MCP-1, MIP-1α and β, RANTES, TNF-α, IL-1β and IL-6 from the mucosa. SP (0.1 μM) had comparable actions, but had no effect on MCP-1 or RANTES. These effects were inhibited separately by tachykinin NK 1 and NK 2 receptor antagonists SR140333 and SR48968 (both 0.1 μM), suggesting that these responses were mediated by both NK1 and NK2 receptors. In conclusion, these data support a novel inflammatory role for HK-1 in human colon, signaling via NK1 and NK2 receptors (and possibly other tachykinin-preferring receptors) to regulate the release of a broad spectrum of proinflammatory mediators. The study suggests that along with SP, HK-1 is also a proinflammatory mediator, likely involved in colonic inflammation, including inflammatory bowel disease (IBD)., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: The tachykinin family of peptides (substance P, neurokinin A) via the neurokinin-1 (NK-1), NK-2, and NK-3 receptors is involved in many physiological/physiopathological actions. Antagonists of these receptors may be used to treat many human pathologies., Areas Covered: This review offers an overview (from 2014 to present) of the actions exerted by NK receptor (NK-R) antagonists on emesis, pruritus, cardiomyopathy, respiratory tract diseases, bacterial infection, cancer, ocular pain, corneal neovascularization, excess of body fat/weight, conditioned fear, social isolation stress, hot flush, melanogenesis, follicle development, fish reproduction, and sex-hormone-dependent diseases., Expert Opinion: From 2014, no invention has been published using NK-2R antagonists. Although the tachykinin/NK receptor system is involved in a great number of mechanisms, to date, the use of only five NK-1R antagonists have been approved in humans but no NK-2R or NK-3R antagonist. NK receptor antagonists are safe in human trials and are potential therapeutic agents, but this potential is currently minimized. In humans, more studies on molecules acting as NK receptor antagonists and exerting a potential therapeutic action must be carried out. The antipruritic or antitumor action of NK-1R antagonists must be explored in greater depth: the highest safe dose and the time of administration (for a long period of time) of these antagonists must be well established.

In pigs, plasma prolactin concentration markedly changes during the oestrous cycle and the regulation of its secretion is very complex. The contribution of neurokinins in this process has not been sufficiently delineated. The aim of the study was to examine the effects of neurokinin A (NKA) on prolactin synthesis and secretion in cyclic gilts. The expression of NKA precursor (Ppta) and receptor (Tacr2) genes as well as NKA and TACR proteins content in the porcine pituitaries (days 2-3, 9-10, 12-13, 15-16 and 19-20 of the cycle) was determined. Furthermore, the in vitro influence of NKA on the expression of prolactin (Prl), dopamine receptor (D2r), TRH receptor (Trhr) genes and prolactin secretion by the porcine pituitary cells (days 9-10, 15-16 and 19-20 of the cycle) was assessed. The expression of Ppta and Tacr2 as well as NKA and TACR proteins in the pituitary tissue has been changing throughout the oestrous cycle. NKA affected in vitro the expression of studied genes and prolactin secretion depending on the stage of the cycle, dose of NKA and/or duration of the cell incubation. Altogether, the study indicates that NKA is engaged in the modulation of prolactin secretion in the pig during the oestrous cycle., (© 2020 Blackwell Verlag GmbH.)

Acute administration of [Lys5,Me,Leu9,Nle10]-NKA(4-10) (LMN-NKA) produces contractions of the detrusor and rectum with voiding in intact and acutely spinal cord injured (SCI) rats. In the current study, the ability of LMN-NKA (10 μg/kg or 100 μg/kg, subcutaneous [SC], twice a day [bid]) or vehicle to induce voiding and defecation in chronic SCI rats was examined across 30 days. After the last day of administration, voiding response rates and bladder pressure (BP) responses to LMN-NKA (intravenous [IV] and SC) were evaluated under anesthesia. In conscious rats, LMN-NKA (100 μg/kg) produced dose-dependent micturition within 5 min, with response rates >90%, and voiding efficiency >80% in males and >60% in females, which remained stable across the 1-month test period. Similarly, LMN-NKA administration rapidly induced defecation, which also remained stable. Under anesthesia, LMN-NKA increased BP, voiding efficiency, and voiding response rates, which reached 100% at 3 and 10 μg/kg IV in males and females, respectively. SC administration produced 100% response rates in males (30 μg/kg) but only 71% in females (100 μg/kg). Efficacy in rats chronically treated with LMN-NKA was similar to naïve and vehicle-treated rats, except for reduced voiding efficiency in chronically dosed female rats (100 μg/kg). No differences in bladder weights or collagen-to-smooth muscle ratios in histological sections were seen between the groups. Thus neither tolerance, nor sensitization, to LMN-NKA-induced micturition and defecation occurs with chronic administration in rats with chronic SCI. Efficacy was higher in male than in female rats.

The tachykinin 2 receptor (NK2R) plays critical roles in gastrointestinal, respiratory and mental disorders and is a well-recognized target for therapeutic intervention. To date, therapeutics targeting NK2R have failed to meet regulatory agency approval due in large part to the limited characterization of the receptor-ligand interaction and downstream signaling. Herein, we report a protein engineering strategy to improve ligand-binding- and signaling-competent human NK2R that enables a yeast-based NK2R signaling platform by creating chimeras utilizing sequences from rat NK2R. We demonstrate that NK2R chimeras incorporating the rat NK2R C-terminus exhibited improved ligand-binding yields and downstream signaling in engineered yeast strains and mammalian cells, where observed yields were better than 4-fold over wild type. This work builds on our previous studies that suggest exchanging the C-termini of related and well-expressed family members may be a general protein engineering strategy to overcome limitations to ligand-binding and signaling-competent G protein-coupled receptor yields in yeast. We expect these efforts to result in NK2R drug candidates with better characterized signaling properties., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The tachykinin neurokinin B (NKB, Tac2) is critical for proper GnRH release in mammals, however, the role of the other tachykinins, such as substance P (SP) and neurokinin A (NKA) in reproduction, is still not well understood. In this study, we demonstrate that NKA controls the timing of puberty onset (similar to NKB and SP) and stimulates LH release in adulthood through NKB-independent (but kisspeptin-dependent) mechanisms in the presence of sex steroids. Furthermore, this is achieved, at least in part, through the autosynaptic activation of Tac1 neurons, which express NK2R (Tacr2), the receptor for NKA. Conversely, in the absence of sex steroids, as observed in ovariectomy, NKA inhibits LH through a mechanism that requires the presence of functional receptors for NKB and dynorphin (NK3R and KOR, respectively). Moreover, the ability of NKA to modulate LH secretion is absent in Kiss1KO mice, suggesting that its action occurs upstream of Kiss1 neurons. Overall, we demonstrate that NKA signaling is a critical component in the central control of reproduction, by contributing to the indirect regulation of kisspeptin release., (Copyright © 2019 Endocrine Society.)

Compounds targeting multiple proteins can have synergistic effects and are therefore of interest in medicinal chemistry. At the same time, inhibiting protein-protein interactions (PPI) is increasingly desired in the treatment of disorders or diseases. The development of non-peptidomimetic inhibitors is still a challenge. Herein we investigate macrocyclic scaffolds with one or two embedded carbohydrates (MECs) that present amino acid side chains, or related isosteres, as pharmacophoric groups. Firstly, retroscreening of the previously reported eannaphane-40 (E40, 40), a MEC presenting two pharmacophoric groups, against a set of 55 receptor-subtypes led to a finding of sub-micromolar inhibitory activity for E40 against three serotonergic isoforms (5HT 1A / 2A/2B ) as well as the Na + channel and the NK-2 receptor. We synthesised MECs with an additional pharmacophoric group compared to E40, with a view to identifying compounds where the selectivity profile was altered among the protein hits from the retroscreening. MECs were produced based on scaffolds with two monosaccharide residues, leading to the incorporation of a third pharmacophoric group. Later, homology models were prepared for four proteins (5HT 1A , 5HT 2A , NK 2 and site-2 of the sodium channel) whose 3D structure is unknown. Inverse docking of the synthesised compounds led to the selection of a new MEC (MEC-B) for protein binding assays. MEC-B was found to have its selectivity profile modulated, in line with docking prediction, compared to E40. MEC-B is dual inhibitor of both 5-HT 1A and the sodium channel with improved selectivity for these proteins compared to 5-HT 2A/2B/2C , 5-HT transporter and NK 2 receptor. Thus, a new multitargeting compound, with an improved selectivity profile was identified, based on a MEC peptidomimetic scaffold., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Tacr2, the gene encoding the NK2 receptor, belongs to G protein-coupled receptors. Accumulating evidence has indicated that the tachykinin receptors may contribute to the pathophysiology of depression. During the last decade, some studies have shown that Tacr2 activation is involved in the modulation of emotional processes. However, the extent, to which stress impacts Tacr2 expression remains unclear. The molecular mechanisms underlying depression also remain poorly understood. In this study, we subjected adult male Sprague Dawley (SD) rats to chronic unpredictable mild stress (CUMS) to induce a depression-like phenotype. We then measured the body weight and performed the sucrose preference test, forced swimming test (FST) and open field test to detect the effects of stress on anhedonia and activity. Western blotting and real-time PCR were used to study the protein and mRNA expression levels of Tacr2, respectively, in the hypothalamus. To explore DNA methylation of the Tacr2 gene, we used methylated DNA immunoprecipitation sequencing (MeDIP-seq). Additionally, we used the bisulfite sequencing PCR (BSP) to further verify the DNA methylation levels of the Tacr2 receptor gene in rats. We found that the CUMS-sensitive rats exhibited a decrease in body weight and sucrose preference, a decrease in the distance traveled, rearing frequency and velocity in the open field test, and an increase in immobility time in the FST. Compared with the expression in the control rats, Tacr2 protein and mRNA expression in the hypothalamus significantly increased in the CUMS-sensitive rats; however, the DNA methylation levels of the Tacr2 gene were significantly lower than in the control rats. In summary, according to our findings, the stress-induced increase in Tacr2 expression in the hypothalamus correlated with a specific decrease in DNA methylation of the Tacr2 gene. These results may enrich the understanding of the pathological processes of depression and provide insights into therapeutic approaches for its treatment., (Copyright © 2019. Published by Elsevier B.V.)

Tachykinins act as neurotransmitters and neuromodulators in the central and peripheral nervous system. Preclinical studies and clinical trials showed that inhibition of the tachykinin receptors, mainly NK2 may constitute a novel attractive option in the treatment of irritable bowel syndrome (IBS). In this review, we focused on the role of tachykinins in physiology and pathophysiology of gastrointestinal (GI) tract. Moreover, we summed up recent data on tachykinin receptor antagonists in the therapy of IBS. Ibodutant is a novel drug with an interesting pharmacological profile, which exerted efficacy in women with diarrhea-predominant IBS (IBS-D) in phase II clinical trials. The promising results were not replicable and confirmed in phase III of clinical trials. Ibodutant is not ready to be introduced in the pharmaceutical market and further studies on alternative NK2 antagonist are needed to make NK2 antagonists useful tools in IBS-D treatment.

Gastric ulceration, a focal tissue damage accompanied by inflammation, can influence other parts of the stomach. Substance P and its receptors are strongly involved in regulation of gastrointestinal motility, secretion and inflammation. The enteric nervous system is one of the regulators of gastrointestinal functioning and contributes to tissue response to the pathology. The pig, an omnivorous animal, is a valuable species for gastrointestinal experiments. Thus, the objective of the study was to verify whether the antral ulceration induces changes in the expression of substance P and tachykinin receptors in the neighboring (antrum) and distanced (corpus, pylorus) porcine gastric tissues and therein localized myenteric and submucosal perikarya as well as in the intrinsic descending neurons supplying pyloric sphincter. The experiment was performed on healthy pigs and pigs with experimentally induced gastric ulcers. Stomach samples from the corpus, antrum (adjacent to the ulcer in experimental pigs) and pylorus were analyzed by: (1) double immunofluorescence for changes in the number of SP-positive myenteric and submucosal neurons (2) Real-Time PCR for changes in expression of mRNA encoding SP and Nk1, Nk2, Nk3 receptors. Additionally, gastric descending neurons supplying pyloric sphincter were immunostained for SP. In experimental animals, only the number of SP-positive myenteric perikarya significantly increased in all stomach localizations studied. Q-PCR revealed increased expression for: SP, Nk1, Nk3 in the corpus; Nk2 and Nk3 in the pylorus; In the antrum, expression of Nk3 was increased but Nk2-decreased. Antral ulcers induced significant changes in the expression of SP and tachykinin receptors in the wide stomach area indicating sophisticated tissue reaction.

Introduction: The biological action of Substance P (SP) is mediated mainly by NK-1 receptors (NK1R) followed by NK2 receptors (NK2R). Aberrant expression of NK1R and NK2R has been identified in various carcinomas. The role of Substance P and its receptors, especially NK2R in cancer progression is not entirely known and there are conflicting results in the literature demonstrating the need for further investigation. In the current study, we examined the effects of SP and antagonists selective for the NK1R and NK2R in breast carcinoma cells metastasize to vital organs., Methods: The effects of highly potent and selective non-peptide mouse NK1R and NK2R antagonists RP 67,580 and GR 159897, respectively, as well as SP and SP methyl ester, on both metastatic (4THM, 4TBM, 4TLM, 4T1) and non-metastatic (67NR) breast cancer cells were determined., Results: NK1R and NK2R were over expressed in metastatic breast cells compared to non-metastatic cells. The NK1R antagonist at a 30 μM dose inhibited cell growth and induced cell death in metastatic cells while enhancing phosphorylation of Akt, the latter response not observed in the non-metastatic 67NR cells. Blocking the action of SP at the NK2R (30 μM antagonist) suppressed cellular proliferation in all the cell lines examined, with a response less prominent than that of the NK1R antagonist. Differently, the NK2R antagonist increased phosphorylation of p38 and enhanced MIP-2 secretion. SP and the SP methyl ester neither altered cell proliferation nor the effects of NK1R and NK2R antagonists in the metastatic cell lines., Conclusions: Increased sensitivity of metastatic breast carcinoma cells to NK1R and NK2R antagonists suggest potential therapeutic value of antagonists in metastatic disease. NK1R and NK2R in metastatic breast carcinoma cells react differently to agonists and antagonists. These findings together with previously published data demonstrate that differential consequences of receptor antagonists and SP may inhibit breast cancer growth and metastasis., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Tachykinins are expressed within bladder-innervating sensory afferents and have been shown to generate detrusor contraction and trigger micturition. The release of tachykinins from these sensory afferents may also activate tachykinin receptors on the urothelium or sensory afferents directly. Here, we investigated the direct and indirect influence of tachykinins on mechanosensation by recording sensory signaling from the bladder during distension, urothelial transmitter release ex vivo, and direct responses to neurokinin A (NKA) on isolated mouse urothelial cells and bladder-innervating DRG neurons. Bath application of NKA induced concentration-dependent increases in bladder-afferent firing and intravesical pressure that were attenuated by nifedipine and by the NK2 receptor antagonist GR159897 (100 nM). Intravesical NKA significantly decreased bladder compliance but had no direct effect on mechanosensitivity to bladder distension (30 µl/min). GR159897 alone enhanced bladder compliance but had no effect on mechanosensation. Intravesical NKA enhanced both the amplitude and frequency of bladder micromotions during distension, which induced significant transient increases in afferent firing, and were abolished by GR159897. NKA increased intracellular calcium levels in primary urothelial cells but not bladder-innervating DRG neurons. Urothelial ATP release during bladder distention was unchanged in the presence of NKA, whereas acetylcholine levels were reduced. NKA-mediated activation of urothelial cells and enhancement of bladder micromotions are novel mechanisms for NK2 receptor-mediated modulation of bladder mechanosensation. These results suggest that NKA influences bladder afferent activity indirectly via changes in detrusor contraction and urothelial mediator release. Direct actions on sensory nerves are unlikely to contribute to the effects of NKA.

The purpose of this study was to investigate if AZD5329, a dual neurokinin NK1/2 receptor antagonist, is a suitable candidate for further development as an oral immediate release (IR) solid dosage form as a final product. The neutral form of AZD5329 has only been isolated as amorphous material. In order to search for a solid material with improved physical and chemical stability and more suitable solid-state properties, a salt screen was performed. Crystalline material of a maleic acid salt and a fumaric acid salt of AZD5329 were obtained. X-ray powder diffractiometry, thermogravimetric analysis, differential scanning calorimetry and dynamic vapor sorption were used to investigate the physicochemical characteristics of the two salts. The fumarate salt of AZD5329 is anhydrous, the crystallization is reproducible and the hygroscopicity is acceptable. Early polymorphism assessment work using slurry technique did not reveal any better crystal modification or crystallinity for the fumarate salt. For the maleate salt, the form isolated originally was found to be a solvate, but an anhydrous form was found in later experiments; by suspension in water or acetone, by drying of the solvate to 100-120 °C or by subjecting the solvate form to conditions of 40 °C/75%RH for 3 months. The dissolution behavior and the chemical stability (in aqueous solutions, formulations and solid-state) of both salts were also studied and found to be satisfactory. The compound displays sensitivity to low pH, and the salt of the maleic acid, which is the stronger acid, shows more degradation during stability studies, in line with this observation. The presented data indicate that the substance fulfils basic requirements for further development of an IR dosage form, based on the characterization on crystalline salts of AZD5329., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tachykinin neurokinin 2 (NK2) receptor agonists may have potential to alleviate clinical conditions associated with bladder and gastrointestinal underactivity by stimulating contraction of visceral smooth muscle. The ability of [Lys 5 ,MeLeu 9 ,Nle 10 ]-neurokinin A (4-10) (LMN-NKA) to elicit micturition and defecation was examined after repeated administration in groups of 2-10 conscious dogs. Administration of 10-100 μ g/kg, i.v., four times daily for six consecutive days, reliably elicited micturition after ≥90% of doses and defecation after ≥50% of doses. Voiding occurred <4 minutes after dosing and was short lasting (<10 minutes). LMN-NKA was well tolerated, with emesis after ∼25% of doses at 100 μ g/kg, i.v. Hypotension was induced by 100 μ g/kg, i.v., of LMN-NKA but not by lower doses. Administration of 30-300 μ g/kg, s.c., twice daily for seven consecutive days, reliably elicited both urination and defecation after 88%-100% of doses, and was accompanied by a high rate of emesis (50%-100%). The onset of voiding was rapid (<7 minutes) but was more prolonged than after intravenous administration (30-60 minutes). Emesis induced by 30 or 300 μ g/kg, s.c., of LMN-NKA was significantly reduced (from 58% to 8% and from 96% to 54%, respectively) by a 30-minute pretreatment with the neurokinin 1 (NK1) receptor antagonist, (2 S ,3 S )- N -(2-methoxybenzyl)-2-phenylpiperidin-3-amine (CP-99,994; 1 mg/kg, s.c.). The ability of selective NK2 receptor agonists to elicit on-demand voiding could potentially address a major unmet need in people lacking voluntary control of micturition and/or defecation. LMN-NKA unexpectedly activated NK1 receptors at doses that stimulated voiding, causing emesis and hypotension that may limit the clinical utility of nonselective NK2 receptor agonists., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Background & Aims: Tachykinins are involved in physiological and pathophysiological mechanisms in the gastrointestinal tract. The major sources of tachykinins in the gut are intrinsic enteric neurons in the enteric nervous system and extrinsic nerve fibers from the dorsal root and vagal ganglia. Although tachykinins are important mediators in the enteric nervous system, how they contribute to neuroinflammation through effects on neurons and glia is not fully understood. Here, we tested the hypothesis that tachykinins contribute to enteric neuroinflammation through mechanisms that involve intercellular neuron-glia signaling., Methods: We used immunohistochemistry and quantitative real-time polymerase chain reaction, and studied cellular activity using transient-receptor potential vanilloid-1 ( TRPV1 ) tm1 (cre)Bbm /J::Polr2a tm1(CAG-GCaMP5g,-tdTomato)Tvrd and Sox10 CreER T2 ::Polr2a tm1(CAG-GCaMP5g,-tdTomato)Tvrd mice or Fluo-4. We used the 2,4-di-nitrobenzene sulfonic acid (DNBS) model of colitis to study neuroinflammation, glial reactivity, and neurogenic contractility. We used Sox10 ::CreER T2+/- / Rpl22 tm1.1Psam /J mice to selectively study glial transcriptional changes., Results: Tachykinins are expressed predominantly by intrinsic neuronal varicosities whereas neurokinin-2 receptors (NK2Rs) are expressed predominantly by enteric neurons and TRPV1-positive neuronal varicosities. Stimulation of NK2Rs drives responses in neuronal varicosities that are propagated to enteric glia and neurons. Antagonizing NK2R signaling enhanced recovery from colitis and prevented the development of reactive gliosis, neuroinflammation, and enhanced neuronal contractions. Inflammation drove changes in enteric glial gene expression and function, and antagonizing NK2R signaling mitigated these changes. Neurokinin A-induced neurodegeneration requires glial connexin-43 hemichannel activity., Conclusions: Our results show that tachykinins drive enteric neuroinflammation through a multicellular cascade involving enteric neurons, TRPV1-positive neuronal varicosities, and enteric glia. Therapies targeting components of this pathway could broadly benefit the treatment of dysmotility and pain after acute inflammation in the intestine.

Background: Tacr2 is one of the G protein-coupled receptors(GPCRs) that mediate the biological actions of tachykinins. It is abundantly expressed in the gastrointestinal (GI) system and is thought to play an important role in GI motility, secretion, and visceral sensitivity. Previously, the physiological and pathophysiological functions of Tacr2 were mainly studied using Tacr2 selective agonists or antagonists. Here, we seek to investigate the effect of Tacr2 disruption in mice to provide further insights., Methods: The Tacr2 knockout mice were generated by homologous recombination and the phenotypic changes of the Tacr2-null mice were analyzed and compared with their wild type (wt) littermates., Key Results: Increased food retention was detected in Tacr2 -/- mice. The stomach of Tacr2 -/- mice had thinner muscularis externa and less neurons in the myenteric plexus. The stomach and small intestine exhibited longer duration of electrical field stimulation (EFS)-induced inhibition in the gastric fundus and decreased frequency of migrating motor complex (MMC), respectively. Neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP) were significantly up-regulated due to Tarc2 deficiency, contributing to enhanced nitric oxide (NO) signaling in the stomach of Tacr2 -/- mice. Intraperitoneal application of 7-nitroindazole (7-NI) to Tacr2 -/- mice effectively relieved the gastric emptying disturbance. Moreover, Creb and NF-κB signalings were involved in the regulation of these physiological changes initiated by Tacr2 deficiency., Conclusions & Inferences: Tacr2 negatively regulated the expression of nNOS and VIP both in vivo and in vitro. Its ablation in mice elevated the expression of nNOS and VIP, enhanced NO signaling and changed the Creb and NF-κB signalings, finally leading to the gastric emptying disturbance of Tacr2 -/- mice., (© 2017 John Wiley & Sons Ltd.)

We evaluated the pharmacological characteristics of KRP-103, chemically named as ((2-(4-acetylpiperazin-1-yl)-6-[3,5-bis(trifluorometheyl)-phenylmethyl]-4- (2-methylphenyl)-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,5]oxazocin-5-one), and its effects on lower urinary tract function in guinea pigs. In radioligand binding assay, KRP-103 showed higher selectivity for human NK1 receptor (hNK1R) than for human NK2 receptor (hNK2R) and human NK3 receptor (hNK3R) (Ki for hNK1R, hNK2R and hNK3R=0.126 nM, > 10 000 nM and > 10 000 nM). In distention-induced rhythmic bladder contractions (RBCs) in urethane-anesthetized guinea pigs, intravenous administration of KRP-103 dose-dependently increased the shutdown time of RBCs and slightly decreased the amplitude of RBCs (only about 20%). In acutely spinalized guinea pig cystometory, intraduodenal administration of KRP-103 dose-dependently increased the effective bladder capacity with a minimum effective dose of 1 mg/kg. Furthermore, to clarify the site of action of KRP-103, we evaluated the inhibitory effects of KRP-103 on bladder contractions induced by electrical stimulation (ES) of the central or peripheral cut end of the pelvic nerve (PN). KRP-103 inhibited the bladder contractions induced by ES of the central cut end of PN but not those induced by ES of the peripheral cut end of PN. These results indicate that KRP-103 enhances bladder storage function by inhibiting sensory transmission from the bladder at the level of spinal cord without affecting bladder efferent function, suggesting that KRP-103 may be a suitable therapeutic drug for treatment of overactive bladder., Competing Interests: Conflict of Interest: The authors declare no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Duodenal reflux of fluids containing trypsin relates to refractory gastroesophageal reflux disease (GERD). Esophageal peristalsis and clearance are important factors in GERD pathogenesis. However, the function of trypsin in esophageal body contractility is not fully understood. In this study, effects of trypsin on circular smooth muscle (CSM) and longitudinal smooth muscle (LSM) of the porcine esophageal body were examined. Trypsin elicited a concentration dependent biphasic response, a major contraction and a subsequent relaxation only in CSM. In CSM, contraction occurred at trypsin concentrations of 100nM and relaxation at 1μM. A proteinase-activated receptor (PAR)2 activating peptide, SLIGKV-NH 2 (1mM), induced a monophasic contraction. Those responses were unaffected by tetrodotoxin though abolished by the gap junction uncouplers carbenoxolone and octanol. They were also partially inhibited by a transient receptor potential vanilloid type 1 (TRPV1) antagonist and abolished by combination of neurokinin receptor 1 (NK 1 ) and NK 2 antagonists, but not by an NK 3 antagonist, suggesting a PAR2-TRPV1-substance P pathway in sensory neurons. Substance P (100nM), an agonist for various NK receptors (NK 1 , NK 2 and NK 3 ) with differing affinities, induced significant contraction in CSM, but not in LSM. The contraction was also blocked by the combination of NK 1 and NK 2 antagonists, but not by the NK 3 antagonist. Moreover, substance P-induced contractions were unaffected by the TRPV1 antagonist, but inhibited by a gap junction uncoupler. In conclusion, trypsin induced a biphasic response only in CSM and this was mediated by PAR2, TRPV1 and NK 1/2 . Gap junctions were indispensable in this tachykinin-induced response., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The purpose of this study was to determine feasibility of a novel therapeutic approach to drug-induced voiding after spinal cord injury (SCI) using a well-characterized, peptide, neurokinin 2 receptor (NK 2 receptor) agonist, Lys 5 , MeLeu 9 , Nle 10 -NKA (4-10) (LMN-NKA). Cystometry and colorectal pressure measurements were performed in urethane-anesthetized, intact, and acutely spinalized female rats. Bladder pressure and voiding were monitored in response to intravenous LMN-NKA given with the bladder filled to 70% capacity. LMN-NKA (0.1-300 μg/kg) produced dose-dependent, rapid (<60 s), short-duration (<15 min) increases in bladder pressure. In intact rats, doses above 0.3-1 μg/kg induced urine release (voiding efficiency of ~70% at ≥1 μg/kg). In spinalized rats, urine release required higher doses (≥10 μg/kg) and was less efficient (30-50%). LMN-NKA (0.1-100 μg/kg) also produced dose-dependent increases in colorectal pressure. No tachyphylaxis was observed, and the responses were blocked by an NK 2 receptor antagonist (GR159897, 1 mg/kg i.v.). No obvious cardiorespiratory effects were noted. These results suggest that rapid-onset, short-duration, drug-induced voiding is possible in acute spinal and intact rats with intravenous administration of an NK 2 receptor agonist. Future challenges remain in regard to finding alternative routes of administration that produce clinically significant voiding, multiple times per day, in animal models of chronic SCI.

Neurokinin A (NKA) is a peptide neurotransmitter that participates in the regulation of breathing and the cardiovascular system. The purpose of the current study was to determine the cardiorespiratory pattern exerted by the systemic injection of NKA, to look at the contribution of neurokinin NK1 and NK2 receptors, and to establish the engagement of the vagal pathway in mediation of these responses. The effects of intravenous injections of NKA (50 μg/kg) were studied in anaesthetized, spontaneously breathing rats in the following experimental schemes: in neurally intact rats; and vagotomized at either midcervical or supranodosal level. Intravenous injections of NKA in the intact rats evoked sudden and short-lived increase in the respiratory rate concomitant with drop in tidal volume, followed by a prolonged depression, coupled with continuous augmentation of the tidal volume. Respiratory alterations were accompanied by transient tachycardia and prolonged hypotension. Midcervical vagotomy eliminated respiratory rate response and augmentation of tidal volume. Section of supranodosal vagi abrogated all respiratory reactions. NK2 receptor blockade abolished respiratory changes without affecting cardiovascular effects, whereas NK1 receptor blockade significantly reduced hypotension and increase in heart rate with no impact on the respiratory system. These results indicate that NKA induced changes in the breathing resulting from an excitation of the NK2 receptors on the vagal endings. A fall in blood pressure triggered by NKA occurs outside of the vagus nerve and is probably mediated via its direct action on vascular smooth muscles supplied with NK1 receptors., (© 2016 John Wiley & Sons Australia, Ltd.)

Background and Purpose: The tachykinin NK2 receptor antagonist ibodutant is under Phase III clinical investigation to treat female patients with irritable bowel syndrome. The aim of this study was to investigate the NK2 receptor-related gender specificity in a model of colitis., Experimental Approach: Colitis was induced by rectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS, 0.5 mL, 30 mg·mL(-1) in 30% ethanol) in female and male guinea pigs. Electromyographic recording of the responses to colorectal distension (CRD) was made 3 days later. Ibodutant (0.33 , 0.65, 1.9 and 6.5 mg·kg(-1) ) was given s.c., 30 min before CRD. Release of neurokinin A and substance P from isolated mucosal and smooth muscle tissues following treatment with KCl (80 mM) or capsaicin (10 μM) was measured by EIA. Plasma pharmacokinetics of ibodutant following a single s.c. administration (0.73 or 2.1 mg·kg(-1) ) were measured over 24 h., Key Results: Ibodutant did not affect abdominal contractions in control animals. After TNBS-induced colitis, ibodutant prevented the increased visceral hypersensitivity to CRD in females, at lower doses than in males. Ibodutant pharmacokinetics did not differ between females and males. Tachykinins release was greater in smooth muscle than in mucosal samples. Capsaicin-stimulated release of tachykinins from inflamed mucosal samples from females was significantly lower than in males., Conclusions and Implications: Ibodutant prevented abdominal nociception in a model of visceral hypersensitivity in guinea pigs with a greater efficacy in females than in males. Our results highlight a gender-related difference in colonic visceral hypersensitivity and mucosal nerve activation., (© 2016 The British Pharmacological Society.)

Background: The mechanism underlying chronic stress-induced gastrointestinal (GI) dysmotility has not been fully elucidated and GI hormones have been indicated playing a role in mediating stress-induced changes in GI motor function., Aims: Our objective was to study the possible role of substance P (SP) in the colonic hypermotility induced by repeated water avoidance stress (WAS) which mimics irritable bowel syndrome., Methods: Male Wistar rats were submitted to WAS or sham WAS (SWAS) (1h/day) for up to 10 consecutive days. Enzyme Immunoassay Kit was used to detect the serum level of SP. The expression of neurokinin-1 receptor (NK1R) was investigated by Immunohistochemistry and Western blotting. The spontaneous contraction of muscle strip was studied in an organ bath system. L-type calcium channel currents (ICa,L) of smooth muscle cells (SMCs) were recorded by whole-cell patch-clamp technique., Results: Fecal pellet expulsion and spontaneous contraction of proximal colon in rats were increased after repeated WAS. The serum level of SP was elevated following WAS. Immunohistochemistry proved the expression of NK1R in mucosa, muscularis and myenteric plexus. Western blotting demonstrated stress-induced up-regulation of NK1R in colon devoid of mucosa and submucosa. Repeated WAS increased the contractile activities of longitudinal muscle and circular muscle strips induced by SP and this effect was reversed by a selective NK1R antagonist. The ICa,L of SMCs in the WAS rats were drastically increased compared to controls after addition of SP., Conclusions: Increased serum SP level and up-regulated NK1R in colon may contribute to stress-induced colonic hypermotility. And L-type calcium channels play a potentially important role in the process of WAS-induced dysmotility., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Prenatal nicotinic exposure (PNE) prolongs bronchopulmonary C-fiber (PCF)-mediated apneic response to intra-atrial bolus injection of capsaicin in rat pups. The relevant mechanisms remain unclear. Pulmonary substance P and adenosine and their receptors (neurokinin-A receptor, NK1R and ADA1 receptor, ADA1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) expressed on PCFs are critical for PCF sensitization and/or activation. Here, we compared substance P and adenosine in BALF and NK1R, ADA1R, and TRPV1 expression in the nodose/jugular (N/J) ganglia (vagal pulmonary C-neurons retrogradely labeled) between Ctrl and PNE pups. We found that PNE failed to change BALF substance P and adenosine content, but significantly upregulated both mRNA and protein TRPV1 and NK1R in the N/J ganglia and only NK1R mRNA in pulmonary C-neurons. To define the role of NK1R in the PNE-induced PCF sensitization, the apneic response to capsaicin (i.v.) without or with pretreatment of SR140333 (a peripheral and selective NK1R antagonist) was compared and the prolonged apnea by PNE significantly shortened by SR140333. To clarify if the PNE-evoked responses depended on action of nicotinic acetylcholine receptors (nAChRs), particularly α7nAChR, mecamylamine or methyllycaconitine (a general nAChR or a selective α7nAChR antagonist) was administrated via another mini-pump over the PNE period. Mecamylamine or methyllycaconitine eliminated the PNE-evoked mRNA and protein responses. Our data suggest that PNE is able to elevate PCF NK1R expression via activation of nAChRs, especially α7nAChR, which likely contributes to sensitize PCFs and prolong the PCF-mediated apneic response to capsaicin., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objectives: Mast cells (MCs) and nerves play an important role in allergic rhinitis (AR), but little is known about their crosstalk in AR. The aim of this study was to investigate MC-nerve interaction in the human nasal mucosa during AR., Methods: The association between MCs and nerves, the expression of neuropeptide receptors (neurokinin 1 receptor [NK1R], neurokinin 2 receptor [NK2R], calcitonin gene-related peptide receptor [CGRPR], and MrgX2) on MCs, and protease-activated receptor 2 (PAR2) and tyrosine receptor kinase A (TrkA) on nerve fibres in the human nasal mucosa were investigated with immunofluorescence and real-time PCR., Results: The association between MCs and nerves was found to be significantly increased, although the numbers of MCs and nerve fibres were unchanged during AR. MCs expressing tryptase-chymase (MCtc) were frequently associated with nerve fibres and these contacts increased significantly in AR. Neuropeptide receptors NK1R, NK2R, and CGRPR were firstly found to be largely localised on MCs. The number of MCs expressing NK1R and NK2R, but not CGRPR, was significantly increased in AR. Interestingly, MCtc mostly expressed these neuropeptide receptors. The newly discovered tachykinin receptor MrgX2 was not expressed on nasal MCs, but was expressed on gland cells and increased in AR. Additionally, tachykinergic nerve fibres were found to express PAR2 or TrkA as receptors for MCs., Conclusions: This study revealed for the first time an increase of MC-nerve association and neuropeptide receptor expression on MCs during AR as well as nerve fibres containing receptors for MCs. These results suggest that targeting or controlling airway sensory nerve function as a modulator of MCs may prevent allergic airway inflammation such as AR., (© 2016 S. Karger AG, Basel.)

Several cytoplasmic proteins that are involved in G protein-coupled receptor signaling cascades are known to translocate to the plasma membrane upon receptor activation, such as beta-arrestin2. Based on this example and in order to identify new cytoplasmic proteins implicated in the ON-and-OFF cycle of G protein-coupled receptor, a live-imaging screen of fluorescently labeled cytoplasmic proteins was performed using translocation criteria. The screening of 193 fluorescently tagged human proteins identified eight proteins that responded to activation of the tachykinin NK2 receptor by a change in their intracellular localization. Previously we have presented the functional characterization of one of these proteins, REDD1, that translocates to the plasma membrane. Here we report the results of the entire screening. The process of cell activation was recorded on videos at different time points and all the videos can be visualized on a dedicated website. The proteins BAIAP3 and BIN1, partially translocated to the plasma membrane upon activation of NK2 receptors. Proteins ARHGAP12 and PKM2 translocated toward membrane blebs. Three proteins that associate with the cytoskeleton were of particular interest : PLEKHH2 rearranged from individual dots located near the cell-substrate adhesion surface into lines of dots. The speriolin-like protein, SPATC1L, redistributed to cell-cell junctions. The Chloride intracellular Channel protein, CLIC2, translocated from actin-enriched plasma membrane bundles to cell-cell junctions upon activation of NK2 receptors. CLIC2, and one of its close paralogs, CLIC4, were further shown to respond with the same translocation pattern to muscarinic M3 and lysophosphatidic LPA receptors. This screen allowed us to identify potential actors in signaling pathways downstream of G protein-coupled receptors and could be scaled-up for high-content screening., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Recent evidence suggests that neurokinin B (NKB), a member of the neurokinin (tachykinin) peptide family, plays a pivotal role in gonadotropin-releasing hormone (GnRH) pulse generation. Three types of neurokinin receptors (NKRs), NK1R, NK2R and NK3R, are found in the brain. Although NKB preferentially binds to NK3R, other NKRs are possibly also involved in NKB action. The present study examined the effects of intravenous administration of the NKR subtype-selective agonists GR73632 (NK1R), GR64349 (NK2R), and senktide (NK3R) on GnRH pulse generator activity and luteinizing hormone (LH) secretion. Multiple-unit activity (MUA) was monitored in ovariectomized goats (n = 5) implanted with recording electrodes. Characteristic increases in MUA (MUA volleys) were considered GnRH pulse generator activity. Although three NKR agonists dose-dependently induced an MUA volley and an accompanying increase in LH secretion, the efficacy in inducing the volley markedly differed. As little as 10 nmol of senktide induced an MUA volley in all goats, whereas a dose of 1000 nmol was only effective for the NK1R and NK2R agonists in two and four goats, respectively. When the treatment failed to evoke an MUA volley, no apparent change was observed in the MUA or LH secretion. Similar effects of the NK2R and NK3R agonists were observed in the presence of estradiol. The results demonstrated that NK3R plays a predominant role in GnRH pulse generation and suggested that the contributions of NK1R and NK2R to this mechanism may be few, if any, in goats.

5-Hydroxytryptamine (5-HT) released from colonic mucosal enterochromaffin (EC) cells is a major signaling molecule, which participates in the pathophysiological regulation of colonic functions in gut disorder including irritable bowel syndrome (IBS), but the endogenous modulator system for the 5-HT release is not yet well elucidated. Our in vitro studies in guinea-pig colon have indicated that the cascade pathway of neuronal tachykinergic NK3 receptors and NK2 receptors on peptide YY (PYY)-containing endocrine L cells represents an endogenous modulator system for 5-HT release from EC cells and that melatonin, endogenous tachykinins and PYY play important roles in modulation of the release of 5-HT from EC cells via the endogenous NK2/NK3 receptor cascade system. This review aims at examining the potential role of the endogenous tachykinergic NK2/NK3 receptor cascade system controlling the release of 5-HT from EC cells, with special attention being paid to the pathophysiology of gut disorders including IBS.

The NK3 receptor is a GPCR that is prominently expressed in limbic areas of the brain, many of which have been implicated in schizophrenia. Phase II clinical trials in schizophrenia with two selective NK3 antagonists (osanetant and talnetant) have demonstrated significant improvement in positive symptoms. The objective of this study was to characterize the properties of a novel dual NK2/NK3 antagonist, RO5328673. [(3)H]RO5328673 bound to a single saturable site on hNK2, hNK3 and gpNK3 with high-affinity. RO5328673 acted as an insurmountable antagonist at both human and guinea-pig NK3 receptors in the [(3)H]IP accumulation assay. In binding kinetic analyses, [(3)H]RO5328673 had fast association and dissociation rates at hNK2 while it had a fast association rate and a remarkably slow dissociation rate at gp and hNK3. In electrophysiological recordings of gp SNpc, RO5328673 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurons with an insurmountable mechanism of action. RO5328673 exhibited in-vivo activity in gerbils, robustly reversing the senktide-induced locomotor activity. The TM2 residue gpNK3-A114(2.58) (threonine in all other species) was identified as the critical residue for the RO5328673's slower dissociation kinetics and stronger insurmountable mode of antagonism in the guinea-pig as compared to hNK3-T139(2.58). Using site-directed mutagenesis, [(3)H]RO5328673 binding and rhodopsin-based modeling, the important molecular determinants of the RO5328673-binding pocket of hNK3 were determined. A comparison of the RO5328673-binding pocket with that of osanetant showed that two antagonists have similar contact sides on hNK3 binding crevice except for three mutations V95L(1.42), Y247W(5.38), V255I(5.46), which behaved differently between interacting modes of two antagonists in hNK3., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

TAC3 is a member of tachykinins, and its gene product neurokinin B (NKB) has recently emerged as a key regulator for LH through modulation of kisspeptin/GnRH system within the hypothalamus. In fish models, TAC3 not only encodes NKB but also a novel tachykinin-like peptide called NKB-related peptide (NKBRP), and the pituitary actions of these TAC3 gene products are still unknown. Using grass carp as a model, the direct effects and postreceptor signaling for the 2 TAC3 products were examined at the pituitary level. Grass carp TAC3 was cloned and confirmed to encode NKB and NKBRP similar to that of other fish species. In carp pituitary cells, NKB and NKBRP treatment did not affect LH release and gene expression but up-regulated prolactin (PRL) and somatolactin (SL)α secretion, protein production, and transcript expression. The stimulation by these 2 TAC3 gene products on PRL and SLα release and mRNA levels were mediated by pituitary NK2 and NK3 receptors, respectively. Apparently, NKB- and NKBRP-induced SLα secretion and transcript expression were caused by adenylate cyclase/cAMP/protein kinase A, phospholipase C/inositol 1,4,5-triphosphate/protein kinase C and Ca(2+)/calmodulin/Ca(2+)/calmodulin-dependent protein kinase II activation. The signal transduction for the corresponding responses on PRL release and mRNA expression were also similar, except that the protein kinase C component was not involved. These findings suggest that the 2 TAC3 gene products do not play a role in LH regulation at the pituitary level in carp species but may serve as novel stimulators for PRL and SLα synthesis and secretion via overlapping postreceptor signaling mechanisms coupled to NK2 and NK3 receptors, respectively.

The mTORC1 kinase promotes cell growth in response to growth factors by activation of receptor tyrosine kinase. It is regulated by the cellular energy level and the availability of nutrients. mTORC1 activity is also inhibited by cellular stresses through overexpression of REDD1 (regulated in development and DNA damage responses). We report the identification of REDD1 in a fluorescent live-imaging screen aimed at discovering new proteins implicated in G-protein-coupled receptor signaling, based on translocation criteria. Using a sensitive and quantitative plasma membrane localization assay based on bioluminescent resonance energy transfer, we further show that a panel of endogenously expressed GPCRs, through a Ca(2+)/calmodulin pathway, triggers plasma membrane translocation of REDD1 but not of its homolog REDD2. REDD1 and REDD2 share a conserved mTORC1-inhibitory motif characterized at the functional and structural level and differ most in their N-termini. We show that the N-terminus of REDD1 and its mTORC1-inhibitory motif participate in the GPCR-evoked dynamic interaction of REDD1 with the plasma membrane. We further identify REDD1 as a novel effector in GPCR signaling. We show that fast activation of mTORC1 by GPCRs correlates with fast and maximal translocation of REDD1 to the plasma membrane. Overexpression of functional REDD1 leads to a reduction of mTORC1 activation by GPCRs. By contrast, depletion of endogenous REDD1 protein unleashes mTORC1 activity. Thus, translocation to the plasma membrane appears to be an inactivation mechanism of REDD1 by GPCRs, which probably act by sequestering its functional mTORC1-inhibitory motif that is necessary for plasma membrane targeting.

This Review deals essentially with the elucidation of structural features of Tachykinin family of neuropeptides, which are known to interact through three distinct GPCR subtypes, namely NK1 (Neurokinin 1), NK2 (Neurokinin 2) and NK3 (Neurokinin 3) receptors. In mammals, Tachykinins have been shown to elicit a wide array of activities such as powerful vasodilatation, hypertensive action and stimulation of extravascular smooth muscle and are known to be involved in a variety of clinical conditions including chronic pain, Parkinson's disease, Alzheimer's disease, depression, rheumatoid arthritis, irritable bowel syndrome and asthma. This broad spectrum of action of Tachykinins is attributed to the lack of selectivity of tachykinins to their receptors. All tachykinins interact with all the three-receptor subtypes with SP preferring NK1, NKA preferring NK2 and NKB preferring NK3. This lack of specificity can be accounted for by the conformational flexibility of these short, linear peptides. Hence, identification of structural features of the agonists important for receptor binding and biological activity is of great significance in unraveling the molecular mechanisms involved in tachykinin receptor activation and also in rational design of novel therapeutic agents. Understanding structure of the ligand-receptor complex and analysis of topography of the binding pocket of the tachykinin receptor is also crucial in rational design of drugs.

Kisspeptin neurons located in the arcuate nucleus (ARN) coexpress dynorphin and neurokinin B (NKB) and may interact to influence gonadotropin secretion. Using a kisspeptin-green fluorescent protein mouse model, the present study examined whether the neuropeptides kisspeptin, dynorphin, and NKB modulate the electrical activity of ARN kisspeptin neurons in the adult male mouse. Cell-attached recordings showed that kisspeptin itself had no effect on kisspeptin neuron firing. Dynorphin and the κ-opioid receptor agonist U50-488 evoked a potent suppression of all ARN kisspeptin neuron firing that was blocked completely by the κ-opioid receptor antagonist nor-Binaltorphimine. Both NKB and Senktide, a neurokinin 3 receptor agonist, exerted a potent stimulatory action on ∼95% of ARN kisspeptin neurons. Although the selective neurokinin 3 receptor antagonists SB222200 and SR142801 blocked the effects of Senktide on kisspeptin neurons, they surprisingly had no effect on NKB activation of firing. Studies with selective neurokinin 1 receptor (SDZ-NKT343) and neurokinin 2 receptor (GR94800) antagonists revealed that the activation of kisspeptin neurons by NKB was only blocked completely by a cocktail of antagonists against all 3 tachykinin receptors. Whole-cell recordings revealed that individual kisspeptin neurons were activated directly by all 3 tachykinins substance, P, neurokinin A, and NKB. These experiments show that dynorphin and NKB have opposing actions on the electrical activity of kisspeptin neurons supporting the existence of an interconnected network of kisspeptin neurons in the ARN. However, the effects of NKB result from an unexpected activation of multiple tachykinin receptors.

These experiments were undertaken to assess the mechanisms underlying the antidepressant-like effects of the neurokinin-2 (NK(2)) receptor antagonist saredutant (SR48968) in rats tested in the forced swim test (FST), by analysing hippocampal brain-derived neurotrophic factor (BDNF) and plasma corticosterone [as index of hypothalamic-pituitary-adrenal (HPA) axis activity]. Male Wistar rats received three intraperitoneal injections over 24 h of vehicle, saredutant (5 mg/kg), citalopram (15 mg/kg), clomipramine (50 mg/kg). Rats were subjected to restraint stress (4 h) 24 h prior to the FST procedure. This stress procedure increased immobility and decreased swimming behaviour in the FST; furthermore, it lowered hippocampal BDNF protein expression and increased plasma corticosterone levels. Saredutant and clomipramine or citalopram, used here as positive controls, reduced the immobility time in the FST both under basal conditions and after stress exposure. This effect was not attributable to changes in locomotion, because locomotor activity was unchanged when assessed in the open field test. Pretreatment with para-cholorophenylalanine (150 mg/kg, 72 h and 48 h prior to FST) abolished the effect of citalopram and saredutant on immobility time. At neurochemical level, saredutant attenuated activation of HPA axis in stressed animals more than clomipramine or citalopram. The behavioural effects of saredutant support the hypothesis that NK(2) receptor activity is involved in stress-related disorders. These effects of saredutant may be related to normalization of the HPA axis. Moreover, saredutant increases BDNF expression in the hippocampus, confirming the role of NK(2) receptor blockade in BDNF activation following stressor application.

Increasing evidence suggests that tachykinins are involved in the control of different pathological conditions, including psychiatric disorders. In this study we evaluated the expression of NK(1) and NK(2) receptors (NK-1R and NK-2R), as well as the effects of substance P (SP) and neurokinin A (NKA), in monocytes isolated from 15 healthy subjects and 15 patients with recurrent major depressive disorder (RMDD), under stable antidepressant therapy. NK-1R expression in monocytes from RMDD patients was significantly decreased as compared to healthy subjects, whereas NK-2R expression was markedly increased. Both NK-1R and NK-2R expression correlated with HAM-D, but not HAM-A, score. SP, NKA and selective NK-1R and NK-2R agonists stimulated TNF-α release in monocytes of both groups, with a significant higher effect observed in RMDD. Moreover they induced NF-κB activation, which was reversed by selective NK-1R and NK-2R antagonists, so demonstrating that it was receptor-mediated. The occurrence of a profound alteration in NK receptor expression in RMDD is a novel finding that suggests NK-1R and NK-2R pathways as possible relevant players in major depressive disorder, so improving our understanding of the complex pathogenesis of the disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background and Purpose: The colon-derived peptide hormone, peptide YY (PYY), regulates colonic motility, secretion and postprandial satiety; but little is known about the influence of endogenous PYY on 5-HT release from colonic mucosa. Tachykinin NK(2) receptor-selective agonist, βAla-NKA-(4-10) induces 5-HT release from guinea pig colonic mucosa via NK(2) receptors on the mucosal layer. The present study was designed to determine the influence of endogenous PYY on 5-HT release from guinea pig colonic mucosa, evoked by the NK(2) receptor agonist, βAla-NKA-(4-10)., Experimental Approach: Muscle layer-free mucosal preparations of guinea pig colon were incubated in vitro and the outflow of PYY or 5-HT and its metabolite, 5-HIAA, from these preparations were determined by enzyme immunoassays or HPLC with electrochemical detection respectively., Key Results: βAla-NKA-(4-10) produced a tetrodotoxin-resistant sustained increase in the outflow of PYY and 5-HT from the mucosal preparations. The βAla-NKA-(4-10)-evoked 5-HT outflow was partially inhibited by Y(1) receptor antagonist, BIBO3304, and Y(2) receptor antagonist, BIIE0246, but with less potency. Exogenously-applied PYY also produced a sustained increase in the outflow of 5-HT that was inhibited by Y(1) blockade but not Y(2) blockade., Conclusion and Implications: Our findings support the view that the NK(2) receptor-selective agonist, βAla-NKA-(4-10) produces a long-lasting PYY release from guinea pig colonic mucosa via NK(2) receptors on L cells and βAla-NKA-(4-10)-evoked 5-HT release is in part mediated by endogenously released PYY, acting mainly on Y(1) receptors on EC cells. The PYY-containing L cells appear to play a role in controlling the release of 5-HT from colonic EC cells., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

The neuromodulatory action of the tachykinin NK(3)-receptor agonist [MePhe(7)]-neurokinin B ([MePhe(7)]-NKB) was evaluated on vagal stimulation-induced bronchoconstriction in nonsensitized nonchallenged and ovalbumin (OVA)-sensitized and -challenged guinea pig using the isolated perfused lung preparation. Lungs were placed inside a warmed (37°C) glass chamber and suspended from a force displacement transducer (Grass FT-03) with both vagi connected to a stimulating electrode. Isolated lungs were stimulated at a constant voltage (20 V) and pulse duration (5 ms) with electrical stimulation frequencies ranging from 1 to 128 Hz. The authors demonstrated that vagal stimulation produced frequency-dependent bronchoconstriction and [MePhe(7)]-NKB, at a dose (0.1 μM) that does not produce bronchoconstriction by itself, potentiated the vagally induced bronchoconstriction at all frequencies in nonsensitized nonchallenged animals and to a greater extent in OVA-sensitized and -challenged guinea pigs; the potentiations were totally inhibited by the tachykinin NK(3)-receptor antagonist SR 142801 (1 μM). In a second set of experiments, [MePhe(7)]-NKB produced bronchoconstriction in a dose-dependent (1 to 300 μg/mL) manner with similar potencies and maximum responses in nonsensitized nonchallenged (EC(50) = 8.6 ± 1.1 μM; E(Max) = 61.1 ± 3.5 mm Hg) and OVA-sensitized and -challenged (EC(50) = 8.5 ± 1.3 μM; E(Max) = 63.5 ± 3.7 mm Hg) animals. In conclusion, these results demonstrated that [MePhe(7)]-NKB potentiated vagal stimulation-induced bronchoconstriction via the tachykinin NK(3)-receptors and OVA sensitization caused development of airway hyperresponsiveness in these potentiations. However, OVA sensitization had no effect on airway responsiveness of vagal stimulation-and [MePhe(7)]-NKB-induced bronchoconstrictions.

Substance P is ubiquitous undecapeptide belonging to the tachykinins family. It has been found in the hypothalamus and is involved in the hypothalamo-hypophysial axis in several mammals, including human. Previous studies have shown that substance P increases GH secretions in rats and human. In this study, we have shown that intravenously infused substance P in sheep caused an increased level of Growth Hormone (GH) and GH-Releasing Hormone (GHRH), and decreased Somatotropin Release Inhibiting Hormone (SRIH) secretions. GH was obtained from peripheral blood. GHRH and SRIH were directly collected from hypophysial portal blood, using a trans-nasal surgery technique in a vigil sheep that allowed accessing to hypothalamo-hypophysial portal vessels. Hormones assays were performed by radioimmunoassay (RIA). Moreover, we showed that substance P-induced GH and GHRH secretion appears to be mediated by NK2 tachykinin receptors, since it is specifically blocked by a non peptidic tachykinin NK2 receptor antagonist (SR48968, Sanofi, Montpellier, France) whereas a non peptidic tachykinin NK1 antagonist (SR140333, Sanofi, Montpellier, France) failed to modify GH and GHRH hormones secretions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The neurokinin receptors (NK-R), NK(2)- and NK(3)-R, have been implicated in behavioral processes, but apparently in opposite ways: while NK(2)-R agonism disrupts memory and has anxiogenic-like action, NK(3) -R agonists facilitate memory and display anxiolytic-like effects. Systemic application of NK(2)-R antagonists block the release of acetylcholine (ACh) in the hippocampus, which is induced by intraseptal administration of the NK(2)-R ligand, neurokinin A (NKA). We investigated the effects of medial septal injection of NKA and a preferred ligand of NK(3)-R, neurokinin B (NKB), on the activity of cholinergic neurons of the basal forebrain and assessed the role of the medial septal NK(2)-R in the control of extracellular ACh levels in cholinergic projection areas. ACh was dialysed in the frontal cortex, amygdala and hippocampus of anesthetized animals and was analysed by HPLC-EC. ACh levels in hippocampus and amygdala, but not in frontal cortex were increased after intraseptal injection of either NKA or NKB (0.1, 1, 10 μM). Application of the nonpeptidic NK(2)-R antagonist, saredutant SR48968 (1, 10, 100 pM), followed by NKA (1 μM) or NKB (10 μM) injection into the medial septum, blocked the ACh increase in hippocampus and amygdala. These results indicate that medial septal NK(2)-R have an important role in mediating ACh release, for one, via the septal-hippocampal cholinergic projection and, secondly, via direct or indirect route to the amygdala, but not frontal cortex. They also support the hypothesis that hippocampal cholinergic neurotransmission controls amygdala function suggesting that this interaction is regulated via NK(2)-R in the medial septum., (Copyright © 2010 Wiley Periodicals, Inc.)

The adoptive transfer of OVA-specific Th1 cells into WT mice followed by OVA inhalation induces a significant elevation of airway hyper-responsiveness (AHR) with neutrophilia but not mucus hypersecretion. Here, we demonstrate that the airway inflammation model, pathogenically characterized as severe asthma, was partly mimicked by i.n. administration of IFN-γ. The administration of IFN-γ instead of Th1 cells caused AHR elevation but not neutrophilia, and remarkably induced neurokinin-2 receptor (NK2R) expression along with neurokinin A (NKA) production in the lung. To evaluate whether NKA/NK2R was involved in airway inflammation, we first investigated the role of NKA/NK2R-signaling in airway smooth muscle cells (ASMCs) in vitro. NK2R mRNA expression was significantly augmented in tracheal tube-derived ASMCs of WT mice but not STAT-1(-/-) mice after stimulation with IFN-γ. In addition, methacholine-mediated Ca(2+) influx into the ASMCs was significantly reduced in the presence of NK2R antagonist. Moreover, the NK2R antagonist strongly inhibited IFN-γ-dependent AHR elevation in vivo. Thus, these results demonstrated that IFN-γ directly acts on ASMCs to elevate AHR via the NKA/NK2R-signaling cascade. Our present findings suggested that NK2R-mediated neuro-immuno crosstalk would be a promising target for developing novel drugs in Th1-cell-mediated airway inflammation, including severe asthma., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The present study investigated a role of tachykinins (TK) and neurokinin (NK) receptors (NK-R) in the non-cholinergic regulation of omasal contractions in sheep. Semiquantitative reverse transcription (RT)-PCR revealed that both preprotachykinin (PPT)-A and PPT-B mRNA were distributed in the omasal muscle layers and that NK-R type-1 (NK-1R) and type-2 (NK-2R) mRNA were largely expressed in the same tissues. Cumulative application of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) at 0.03-10μM induced tonic contractions of omasal muscle strips, and the contractile amplitude increased in order of NKB

The tachykinins substance P (SP) and neurokinin A (NKA) are present in nociceptive sensory fibers expressing transient receptor potential cation channel, subfamily V, member 1 (TRPV1). These fibers are found extensively in and around the taste buds of several species. Tachykinins are released from nociceptive fibers by irritants such as capsaicin, the active compound found in chili peppers commonly associated with the sensation of spiciness. Using real-time Ca(2+)-imaging on isolated taste cells, it was observed that SP induces Ca(2+) -responses in a subset of taste cells at concentrations in the low nanomolar range. These responses were reversibly inhibited by blocking the SP receptor NK-1R. NKA also induced Ca(2+)-responses in a subset of taste cells, but only at concentrations in the high nanomolar range. These responses were only partially inhibited by blocking the NKA receptor NK-2R, and were also inhibited by blocking NK-1R indicating that NKA is only active in taste cells at concentrations that activate both receptors. In addition, it was determined that tachykinin signaling in taste cells requires Ca(2+)-release from endoplasmic reticulum stores. RT-PCR analysis further confirmed that mouse taste buds express NK-1R and NK-2R. Using Ca(2+)-imaging and single cell RT-PCR, it was determined that the majority of tachykinin-responsive taste cells were Type I (Glial-like) and umami-responsive Type II (Receptor) cells. Importantly, stimulating NK-1R had an additive effect on Ca(2+) responses evoked by umami stimuli in Type II (Receptor) cells. This data indicates that tachykinin release from nociceptive sensory fibers in and around taste buds may enhance umami and other taste modalities, providing a possible mechanism for the increased palatability of spicy foods.

Clonidine is an α(2)-adrenergic agonist, historically known to treat high blood pressure. Further studies showed that it could be used in the treatment of neuropsychiatric disorders. Afterwards, it has been reported that clonidine stimulated growth hormone (GH) release in many species including man. Using a transnasal surgery technique in awake sheep that allowed accessing hypothalamopituitary portal vessels, our laboratory previously reported that the injection of clonidine in sheep induced a significant, immediate and short-lasting increase in peripheral GH and portal GH-releasing hormone (GHRH) levels. In this study, we show that the clonidine-induced peripheral GH and portal GHRH increase in sheep appears to be mediated by the tachykinin NK2 receptor., (Copyright © 2012 S. Karger AG, Basel.)